Cultivation of Haematococcus pluvialis for astaxanthin production on angled bench-scale and large-scale biofilm-based photobioreactors

The green microalga, Haematococcus pluvialis, is currently cultivated for natural astaxanthin in suspended systems. Immobilised cultivation in a twinlayer (TL) porous substrate bioreactor is a potential revolution in microalgal biotechnology worldwide. For the first time in Vietnam, small-scale (0.05 m2 ) and large-scale (2 m2 ) biofilm-based photobioreactor systems arranged at an angle of 150 were successfully designed, assembled, and operated; the temperature, humidity, air, and light conditions for H. pluvialis cultivation were successfully controlled. Studies were conducted of both systems to determine the optimal storage time of algae after harvest from suspension before inoculation into the TL system, carbon dioxide supply method, light intensity, and initial cell density. In the 0.05 m2 and 2 m2 systems, dry biomass productivity reached 12 g m-2 d-1 (3% astaxanthin content in the dry biomass) and 11.25 g m-2 d-1 (2.8% astaxanthin) after 10 days of cultivation. The 2 m2 biofilm-based photobioreactor system provides many advantages in scaling up astaxanthin production from H. pluvialis.

Life Sciences | Biotechnology Doi: 10.31276/VJSTE.61(3).61-70 Cultivation of Haematococcus pluvialis for astaxanthin production on angled bench-scale and large-scale biofilm-based photobioreactors Hoang-Dung Tran1*, Thanh-Tri Do2, Tuan-Loc Le1, Minh-Ly Tran Nguyen3, Cong-Hoat Pham4, Michael Melkonian5 Nguyen Tat Thanh University, Vietnam Ho Chi Minh city University of Education, Vietnam Vietnam-United States-Australia Biotech Company Limited Minsitry of Sciences and Technology, Vietnam Univeristy of Cologne, Germany Received 10 May 2019; accepted 29 August 2019 Abstract: The green microalga, Haematococcus pluvialis, is currently cultivated for natural astaxanthin in suspended systems Immobilised cultivation in a twinlayer (TL) porous substrate bioreactor is a potential revolution in microalgal biotechnology worldwide For the first time in Vietnam, small-scale (0.05 m2) and large-scale (2 m2) biofilm-based photobioreactor systems arranged at an angle of 150 were successfully designed, assembled, and operated; the temperature, humidity, air, and light conditions for H pluvialis cultivation were successfully controlled Studies were conducted of both systems to determine the optimal storage time of algae after harvest from suspension before inoculation into the TL system, carbon dioxide supply method, light intensity, and initial cell density In the 0.05 m2 and m2 systems, dry biomass productivity reached 12 g m-2 d-1 (3% astaxanthin content in the dry biomass) and 11.25 g m-2 d-1 (2.8% astaxanthin) after 10 days of cultivation The m2 biofilm-based photobioreactor system provides many advantages in scaling up astaxanthin production from H pluvialis Keywords: astaxanthin production, biofilm-based photobioreactor, Haematococcus pluvialis, twin-layer porous, twin-layer system Astaxanthin from H pluvialis and algae suspended cultivation for astaxanthin harvest Astaxanthin is a keto-carotenoid that is mainly used as a supplementary pigment in feedstock for salmon and shrimp cultivation feedstock; it is sometimes also applied in poultry farming to implant colouration in egg yolks [1] Recent studies have shown the strong anti-oxidant activity of astaxanthin in a rat model [2] with benefits to the immune system, cardiac muscles, reducing risks of various cancers, and human skin-ageing treatments [3-8] The green alga H pluvialis is the most common natural astaxanthin producer at the commercial scale This alga species is able to accumulate astaxanthin pigment up to 5.9% of its dry biomass [1, 9, 10] The H pluvialis life cycle includes one biflagellate green cell stage, one nonmotile green cell (palmella) stage, and one thick-walled cyst The green alga(Fig H pluvialis is the most natural astaxanthin (akinete) stage 1) Changes incommon cell states are driven producer at the commercial scale This alga species is able to accumulate astaxanthin pigment up to by environmental conditions The most notable life-history 5.9% of its dry biomass [1, 9, 10] The H pluvialis life cycle includes one biflagellate stage of H pluvialis is the cyst-forming period with its green cell stage, one non-motile green cell (palmella) stage, and one thick-walled cyst distinctive and increase (akinete) stagecell (Fig enlargement 1) Changes in cell states are drivenofbyastaxanthin environmental conditions The most notable life-history stage H pluvialis the cyst-forming period with its production which causes theof change in isalgal color from distinctive cell enlargement and increase of astaxanthin production which causes the green to red [11] change in algal color from green to red [11] Classification number: 3.5 (A) (B) Fig of different H pluvialis life stages: Two-flagellated Fig Microscope Microscopeimage image of different H pluvialis life (A) stages: cells;Two-flagellated (B) Immobilized green thickened wallgreen red cysts (x40) (A) cells;cells (B)and Immobilized cells and thickened wall red cysts (x40) production, H pluvialis is mainly cultured in twoTo attain maximal astaxanthin *Corresponding author: Email: thdung@ntt.edu.vn phase cultivation systems The first phase, known as the green phase or growth phase, is optimised for vegetative growth to achieve a high cell density In suspended cultivation, a maximum light intensity of 150 µmol photon s m-2 s-1 should not be exceeded in order to maintain cell growth and divisions, and environmental parameters such as temperature, carbon dioxide (CO2) levels, and pH need to be closely monitored [1, 12] As the required biomass is attained, the second phase, known as the stressed or red phase, is switched on to stimulate astaxanthin Vietnam Journalaccumulation of Science, [1, 12] September 2019 • Vol.61 Number 61 In the two-phase system, each growth requires different cultivation Technology andphase Engineering conditions and technologies, high energy consumption, and prolonged cultivation time [13, 14] Life Sciences | Biotechnology To attain maximal astaxanthin production, H pluvialis is mainly cultured in two-phase cultivation systems The first phase, known as the green phase or growth phase, is optimised for vegetative growth to achieve a high cell density In suspended cultivation, a maximum light intensity of 150 µmol photons m-2 s-1 should not be exceeded in order to maintain cell growth and divisions, and environmental parameters such as temperature, carbon dioxide (CO2) levels, and pH need to be closely monitored [1, 12] As the required biomass is attained, the second phase, known as the stressed or red phase, is switched on to stimulate astaxanthin accumulation [1, 12] In the two-phase system, each growth phase requires different cultivation conditions and technologies, high energy consumption, and prolonged cultivation time [13, 14] Currently, suspended cultivation of H pluvialis is more common for the production of astaxanthin at the commercial scale Suspended cultivation is applied in open ponds or closed photobioreactors Open-pond cultivation is utilised only for the stressed phase with a short cultivation time (4-6 days) to minimise contamination and apply stressed conditions [12] The closed photobioreactor can minimise contamination and control culture parameters better but it has drawbacks such as of high assembly and maintenance cost [15-17] Moreover, suspended systems have very low biomass concentration (0.05-0.06% of cultivated liquid) and the harvest of algae thus demands additional costs of energy and labour [18] Previous studies of astaxanthin accumulation in H pluvialis in Vietnam: studies of H pluvialis and astaxanthin production in Vietnam have just been conducted since 2010 The Institute of Biotechnology (Vietnam) managed to select one H pluvialis HB strain (own isolate) with a high astaxanthin accumulation capability (4.8% in dry biomass) This strain’s favourable growth conditions include RM culture medium [19], a temperature of 250C, light intensity of 30 µmol photon m-2 s-1, and nitrate as a nitrogen source [20] A maximum cell density of 4.02×106 cells ml-1 was obtained by increasing the nitrate concentration in the RM medium four-fold and switching the light cycle from 12 light/12 dark hours to 16 light/8 dark hours with nutrient supply by exchange of the culture medium [21, 22] To stimulate astaxanthin accumulation, other than the limited nutrient condition, it is important to note that the carbon source is a limiting factor in H pluvialis astaxanthin synthesis [23] With supplementation with 100 mM bicarbonate, the HB strain switched to the cyst stage 62 Vietnam Journal of Science, Technology and Engineering within days and accumulated astaxanthin amounting to 3.96% in the dry biomass [23]; however, this experiment was only conducted at the scale of a 500 ml conical flask containing 350 ml algae liquid cultivated in two separate phases, with sedimentation by gravity and centrifugation to harvest the algal biomass Cultivation at the 10 l scale resulted in an increase in cell density (4.12×106 cells ml-1) though astaxanthin synthesis at this scale has not been investigated [23] Trinh, et al (2017) [24] recently conducted a study using two-phase suspended cultivation In the algal growth phase, the algal cell density increased by only 3.5 times (from an initial density of 2.105 cells ml-1) after 18 days of cultivation In the astaxanthin synthesis induction phase in a l culture medium bioreactor, cell density did not increase after 10 days of cultivation and the astaxanthin content was very low (194 µg l-1) At a larger scale, there are studies using two-phase suspended cultivation in closed systems of 26, 50, and 100 l with a long cultivation period (~25 days) and a relatively complicated process involving multiple centrifugations to increase algal density and exchange the culture medium [21, 22] In the 50 and 100 l systems, the cell density did not improve significantly and there was no report of the astaxanthin content in the dry biomass Immobilised cultivation of H pluvialis in a vertical TL biofilm photobioreactor The TL biofilm photobioreactor was invented by Melkonian and coworkers in Cologne [25, 26] for microalgae biomass cultivation This system is able to hold eight twinlayered modular units (each with a ground size of m2) The algae growth area is 2×0.67 m2 for both sides in one unit [27] The twin-layered structure includes one layer of vertically arranged non-woven glass fiber (80 g m-2, Isola AS Eidanger, Norway) attached to source layers to maintain a continuous medium flow by means of gravity with a flow rate of 6-10 l h-1 m-2 using an agriculture drip-irrigation system (Netafim, Frankfurt, Germany) operating at a maximum pressure of 0.8 bar The prepared culture medium (80-100 l) is stored in closed containers or reservoirs and is distributed all over eight twin-layered structures by two independent pumps (gamma/5b, ProMinent Dosiertechnik GmbH, Germany) After flowing through all these structures, the medium is collected below and directed back into the reservoirs The medium is exchanged once after days [27] Above the source layer a substrate layer is attached by self-adhesion (both layers are hydrophilic) The substrate layer can be made of common printing paper (45-60 g m-2, September 2019 • Vol.61 Number Life Sciences | Biotechnology for instance ‘Kölner Stadt-Anzeiger’, Dumont Schauberg, Cologne, Germany) and is used as carrying agent to immobilise algal cells This substrate layer prevents cells from infliltrating the culture medium and source layer but allows the source layer to control the growth of the immobilised biomass via diffusion of the culture medium [26] Before inoculation into the TL system, algal cells are harvested from the liquid medium by centrifugation at 1,000 g The suspended liquid is inoculated into substrate layers using a paint roller at the density of g dried biomass m-2 The roller is also used to transfer algae from one TL module to another [27] The TL system has been used to to cultivate various algal species, including H pluvialis [10, 25, 28-30] These studies has investigated the influence of many parameters such as the inoculum temperature, light intensity, and nutrient concentration on the immobilised cultivation of H pluvialis; however, these studies were limited by continuous illumination at a maximum intensity of 230 µmol photon m-2 s-1 The immobilised cultivation in these studies was applied the stressed phase of H pluvialis and not to the whole cultivation process, including cell multiplication [10, 28, 30, 31] The TL photobioreactor has recently shown a great promise, achieving production of both biomass and astaxanthin of H pluvialis in only a one-phase system at high light intensity was achieved in a TL photobioreactor recently [32] The algae were cultivated under light intensities ranging from 20 to 1,015 μmol photon m-2 s-1 with 1-10% CO2 added in the gas phase Dried biomass production reached 19.4 g m-2 d-1 and the final dry biomass, 213 g m-2, after 16 days of cultivation During the whole process, the astaxanthin content increased with light intensity and astaxanthin production reached 0.39 g m-2 d-1, with a final amount of astaxanthin of 3.4 g m-2 The astaxanthin content was 2.5% in the dry biomass In comparison with two-phase cultivation using the same TL photobioreactor, one-phase cultivation provided a similar amount of total astaxanthin with half of the cultivation time It was also more convenient than two-phase suspended cultivation [32] Until recently, immobilised cultivation using the TL system included two set-ups: a bench-scale system and a pilot system Both systems are vertically oriented which increased aerial efficiency eight-fold However, the productivity in each unit decreases as mutual shading by the modules decreases the light intensity inside each unit; the investment, maintenance, and harvesting costs also increase per module [27] Immobilised cultivation of H pluvialis on angled a TL biofilm-based photobioreactor for astaxanthin production in Vietnam The use of a vertical biofilm-based photobioreactor for H pluvialis immobilised cultivation in Vietnam involves several difficulties, including higher investment and maintenance costs and the unavailability of several materials (stable non-woven fiberglass and high quality paper) in Vietnam Hanging the modules vertically requires the membranes to be strong enough to withstand gravity the membranes to bearea strong enough withstand gravity The la The larger the surface of the culture,tothe greater the gravity because the mass of the membranes and the water the culture, the greater the gravity because the mass of the me increase Therefore, the vertical system is impractical to increase Therefore, the vertical system is impractical to use use in Vietnam, especially when use of ground area is not when useAccordingly, of groundinarea is nottheanTLissue Accordingly, in Vi an issue Vietnam, biofilm-based solid on a solid photobioreactor should beshould angled be at 15-20 based photobioreactor angled onat a15-20° surface to support the gravity of the membranes gravity of the membranes The bench-scale TL biofilm-based photobioreactor The TL biofilm-based (0 ) forbench-scale H pluvialis immobilised cultivationphotobioreactor includes (0.05 m the following components: supply nutrient immobilised cultivationchamber, includes the system, following components: c circulation system, air circulation system (with or without nutrient circulation system, air circulation system (with or wit CO2), steel frame, and light supply system and light supply system The cultivation chamber is made of acrylic glass because this material allows 90% chamber of light to is be made transmitted (this is glass becau The cultivation of acrylic determined by measuring light intensity before and after it by measurin 90% of light to be transmitted (this is determined passes through the acrylic glass) It is also easy to handle andisafter passes the Each acrylic glass) and more it durable thanthrough silica glass acrylic plate isIt 5is also easy durable thanis attached silica glass Each acrylic plate is mm thic mm thick and via cyanoacrylate glue and sealed by thermal glue Fig presents the technical parameters of presents cyanoacrylate glue and sealed by thermal glue Fig the chamber The cultivation chamber contains supplying of the chamber The cultivation chamber contains supplying el elements for immobilised algae: source layer, substrate layer, algae: source layer, substrate layer,contamination and air conducts Th and air conducts This chamber minimises from the external from environment contamination the external environment Fig Design of bench-scaled system Fig Design of bench-scaled system The provision of nutrients requires a sufficient supply Science, The63dripping nutrie the Number wetness3 ofVietnam the Journal two oflayers Septembermaintain 2019 • Vol.61 Technology and Engineering described in Fig 3A The medium is stored in a 20 l conta system and is continuously pumped into the dripping system Life Sciences | Biotechnology The provision of nutrients requires a sufficient supply of medium liquid to maintain the wetness of the two layers The dripping nutrient irrigation system is described in Fig 3A The medium is stored in a 20 l container located below the system and is continuously pumped into the dripping system via a pumping system with a flow rate of 1.2 l min-1 The dripping system is assembled from a pressurised Capinet dripper with a flow rate of ml min-1, plastic ducts (outer diameter: mm, thickness: mm), and various joints The medium flows through the chamber, wets the layers, and is collected in the reservoir via the duct system Fresh air (with or without a CO2 supplement) is supplied via the system depicted in Fig 3A The main components include a air pump (160 W, 115 l min-1) and an air filte-air is compressed by the pump to a pressure of 0.033 Mpa and flows through the filter The CO2 can be supplemented by air ducts (outer diameter: 10 mm, thickness: mm) leading into the filter; pressurised valves are used to mediate the air pressure to evenly distribute the air to all the chambers Fig indicates the location of the duct system which leads the air into the chambers A steel frame is designed and assembled as indicated in the diagram in Fig 3B The material used is holed 3x3 cm V-shaped steel of mm thickness with an electrostatic coating The components are assembled using bolts and screws designed for holed steel assembly Light system: the experiment utilises many different light sources; the lamps are assembled as show in Fig 3C The lamps are automatically switched on and off by a timer with light cycle of 14 hours light/10 hours dark The light intensity depends on each experiment and was measured using a Lutron LX-1108 (Taiwan) photometer (A) (B) (C) Fig (A) Nutrient and air supply system for cultivation chamber of bench-scale system; (B) Positioning of chambers and lights in Nutrient and for cultivation chamber of bench-scale Fig system;3 (C) (A) The bench-scale system in useair with supply H pluvialis system on the biofilm bench-sca system; (B) Positioning of chambers and lights in bench-scale system; (C) T bench-scale system in use with H pluvialis on the biofilm 64 Vietnam Journal of Science, Fresh air (with or without a CO2 supplement) is supplied via the system depict Technology and Engineering September 2019 • Vol.61 Number in Fig 3A The main components include a air pump (160 W, 115 l min-1) and an a filter - air is compressed by the pump to a pressure of 0.033 Mpa and flows through t Life Sciences | Biotechnology Suitable layer materials for conditions in Vietnam: the materials for algae attachment need to be durable, inexpensive, widely available, and non-toxic; material which can enhance biofilm yield should be preferred Nonwoven fiberglass and printing paper are most often used as a source layer and substrate layer, respectively, in TL photobioreactor for algae cultivation The source layer is made of non-woven fiberglass (0.5x0.1 m) Experiments with substrate layers show that there are only two suitable materials: Whatman filter paper and kraft paper (70 g m-2, Vietnam) These materials are durable with a suitable pore size for keeping the algae in place after immobilisation They were then tested in algae cultivation experiments to compare dry biomass growth in order to select the most appropriate material for use in later studies The results of the H pluvialis cultivation experiment show that dry biomass growth in filter paper and kraft paper is not significantly different (filter paper: 6.81 g m-2 d-1, kraft paper: 6.63 g m-2 d-1, p>0.05) at the same inoculation density of g dry biomass m-2 after 10 days The kraft paper was then selected as the substrate layer since (1) it provides biomass growth similar to that of filter paper, (2) kraft paper is much cheaper than filter paper, (3) kraft paper is widely available in Vietnam, and (4) kraft paper has high physical durability and is easy to handle during cultivation and harvesting (unpublished data) Large-scale biofilm-based photobioreactor (2 m2): in order to scale up the angled TL photobioreactor system, the biotechnology research team of Nguyen Tat Thanh University successfully designed, assembled, and is optimising the angled biofilm-based biophotoreactor for H pluvialis cultivation at a scale of m2 The m2-scaled biofilm-based photobioreactor for H pluvialis immobilised cultivation uses the same component set as the bench-scaled one The large-scale photobioreactor utilises four chambers assembled in the same system; each chamber provides a 0.5 m2 area for algae growth The technical parameters of the large-scale chamber are described in Fig These are the result of several experiments and modifications to suit real-life conditions: (1) Kraft paper and fiberglass plate size of 1x0.6 m; (2) Size and weight of chamber for convenience in handling; (3) Fig (A) Design of large-scale system chamber; (B) Components of the TL photobioreactor system September 2019 • Vol.61 Number Vietnam Journal of Science, Technology and Engineering 65 Life Sciences | Biotechnology A Harwin HP 2500 pump (5-12 W, flow rate: 1.2 l min-1) is used to circulate the medium in the four chambers The duct system is made of soft polyethylene (PE) 16 mm pipes with a 1.2 mm thickness Fresh air (with or without CO2 supplement) is supplied via the system described in Fig The main components are an air pump: 160 W, 115 l min-1; and an air filter: air is compressed by the pump to a pressure of 0.033 Mpa and flows through the filter The CO2 can be supplemented by air ducts (outer diameter: 10 mm, thickness: mm) leading into the filter and pressurised valves The steel frame is designed and assembled as in indicated in the diagram in Fig The material used is holed 3x3 cm V-shaped steel of mm thickness and with electrostatic coating The components are assembled using bolts and screws designed for holed steel assembly The light source for the m2 system includes: (1) a light system that provides 300-1,300 µmol photon m-2 s-1 intensity (provided by eight 400 W Philips high pressure sodium lamps) or (2) a light system that provides 300-1,150 µmol photonm-2 s-1 intensity (provided by ten 250 W Philips high pressure sodium lamps) The lamps are assembled according to Fig The light intensity differed in each experiment and Fig Design of nutrient and air supply system for m2 system chambers was measured using a Lutron LX-1108 (Taiwan) photometer Suitable size to correspond to the light power of the lamps to achieve maximum efficiency; and (4) An appropriate chamber size for manipulation and maintaining a culture below 280C inside the chamber The nutrient supply system is similar to the benchscale system (Fig 5) The large-scale system has its own modifications, for example, a suitable number of drippers in the larger cultivation size (15 drippers/chamber); the drippers are positioned cm away from each other A Harwin HP 2500 pump (5-12 W, flow rate: 1.2 l min-1) is used to circulate the medium in the four chambers The duct system is made of soft polyethylene (PE) 16 mm pipes with a 1.2 mm thickness Fresh air (with or without CO2 supplement) is supplied via the system described in Fig The main components are an air pump: 160 W, 115 l min-1; and an air filter: air is compressed by the pump to a pressure of 0.033 Mpa and flows through the filter The CO2 can be supplemented by air ducts (outer diameter: 10 mm, thickness: mm) leading into the filter and pressurised valves (A) The steel frame is designed and assembled as in indicated in the diagram in Fig The material used is holed 3x3 cm V-shaped steel of mm thickness and with electrostatic coating The components are assembled using bolts and screws designed for holed steel assembly (B) The light source for the m2 system includes: (1) a light system that provides 300-1,300 µmol photon m-2 s-1 Fig (A) Diagram Diagram of ofchamber chamberand andlight lightsource source positioning Fig 6 (A) positioning in 2inm2 2 m2 system; (B) The m system in use with H pluvialis on Thebiofilm m2 system in use with H pluvialis on the biofilm the 66 Vietnam Journal of Science, Technology and Engineering September 2019 • Vol.61 Number system; (B) Life Sciences | Biotechnology intensity (provided by eight 400 W Philips high pressure sodium lamps) or (2) a light system that provides 3001,150 µmol photon m-2 s-1 intensity (provided by ten 250 W Philips high pressure sodium lamps) The lamps are assembled according to Fig The light intensity differed in each experiment and was measured using a Lutron LX1108 (Taiwan) photometer Cultivation of H pluvialis in the astaxanthin accumulation phase on an angled bench-scale TL biofilm-based photobioreactor causes a higher cell death rate and decreases algae growth after immobilisation With an initial density of 7.5 g m-2, average dry biomass production reached 12 g m-2 d-1 after 10 days of cultivation and the astaxanthin content amounted to 3% of the dry biomass (Fig 7) Cultivation of H pluvialis in the astaxanthin accumulation phase on an angled large-scale TL biofilm-based photobioreactor (0.5 m2 x = m2) The experiment was managed to establish the protocol for immobilised H pluvialis cultivation Cultivation of H pluvialis in the astaxanthin accumulation phase onhigh an productivity angled Immobilised algae cultivation for astaxanthin harvest on an angled large-scale system The system is designed bench-scale TL biofilm-based photobioreactor was carried out at bench scale to investigate the factors to maintain a temperature of 24-26 C, and humidity below 80% via a cooling and dehumidifying system to maintain influencing the growth ratecultivation and astaxanthin accumulation Immobilised algae for astaxanthin harvest was carried out at bench algae growth The system operated continuously for 10 days of H pluvialis scale to investigate the factors influencing the growth and dark astaxanthin with 14rate light/10 hours cycle Experiments on the angled 0.05 m bench-scale system accumulation of H pluvialis For experiments on the biofilm, cultures of H pluvialis include: (1) Investigation of the most suitable CO2 supply CCAC 0125 (1) (Culture Collection of Algae at the University system include: Investigation Experiments on theofangled m bench-scale method; (2) Investigation the most0.05 suitable light intensity of Cologne, Germany) were expanded to 10 l PE bags with s-1 were (intensities 200 to µmolmethod; photon (2) m-2 Investigation mostmedium suitable[19] light of the mostfrom suitable CO1,150 supply lofofthe BG11 and placed in 23-250C Algae investigated); (3) Investigation of most suitable initial -2 -1 exposed to a light intensity s were investigated); (3) of 50-60 µmol photons intensity (intensities from 200 to 1,150 µmol photon m were cell density (2.5, 5, 7.5, 10 g dried biomass m-2); and (4) m-2 s-1, a photoperiod of 14/10 Investigationofofthemost suitable initialalgal cellbiomass densitystoring (2.5, 5, 7.5, 10 g dried biomass m-2); hours light/dark cycle and investigation influence of green were aerated with fresh air Microalgae were collected from time on investigation biomass growth andinfluence astaxanthin accumulation and (4) of the of green algal biomass storing timegrowth on biomass the logarithmic phase after 16 days with a Hettich (storing algae at 40C over 1, 3, 5, and days after ROTANA 460 centrifuge (Germany) The percentage growth and astaxanthin accumulation (storing algae at 4°C over 1, 3, 5, and days centrifugation) of flagellate cells after centrifugation was 85%, and the after centrifugation) maximum storage time of the inoculum was 24 hours at 40C At the industrial scale, the inoculum of H pluvialis will be cultured in 80-100 l PE bags The step required to harvest a large number of flagellate cells in suspension is still being solved Initial algae density on biofilm was 5-7.5 g dry biomass m The fixation of algae on biofilm has been tested with many different methods However, using a large brush to fix the algae shows many advantages On average, the time needed to paint m2 of biofilm is minutes The density Fig The microalgae H pluvialis on bench-scale system Fig The microalgae H pluvialis on bench-scale and system 10algae daysareofchecked immediately during qualityafter of the after 10 days of cultivation at an initial dry biomass density of -2 7.5 g m-2 cultivation at an initial dry biomass density of 7.5 g m fixation -2 An appropriate CO2 supply method is aerating fresh TheThe result shows thatthat thethe most supply result shows mostsuitable suitableCOCO method is aerating fresh air into the culture medium to 2 supply air with 1% CO2 supplement supplement into method is aerating fresh air with 1% CO keep pH in 6.5-8.CO The culture medium to supply dissolved to medium used is BG11 [19] with 1% CO2 supplement into the culture and the culture medium to supply dissolved CO2 and to maintain (100 l for 10 days) which is diluted daily to keep electrical maintain a pH favourable for algae growth The most suitable light intensity for dry a pH favourable for algae growth The most suitable light conductivity value in the range of 1,800-2,000 µS cm-2 -2 -1 s providing The mostthe highest biomass growth biomass foranddryastaxanthin is 600-700 isµmolThe photon intensity biomass andaccumulation astaxanthin accumulation light m system -2 -1 The most time and astaxanthin 600-700 photon suitableµmol storing timemis sless than 24suitable hours storing after centrifugation; a longer content storing has timean intensity of 300-800 µmol is less than 24 hours after centrifugation; a longer storing time photons m-2 s-1 causes a higher cell death rate and decreases algae growth after immobilisation With an initial density of 7.5 g m-2, average dry biomass production reached 12 g m-2 d-1 after 10 days of cultivation and the astaxanthin September content amounted to 3% of the dry Vietnam Journal of Science, 2019 • Vol.61 Number Technology and Engineering biomass (Fig 7) 67 Optimisation of high productivity H pluvialis cultivation on a large-scale horizontal system produced some results Average productivity of 11.25 g m-2 d-1 and an astaxanthin content of 2.8% of the dry biomass was obtained from the m2 system in the above-described conditions Contamination was controlled during the cultivation period (Fig 8) The m2 system provided slightly lower yields than the 0.05 m2 system However, astaxanthin productivity was higher in both suspended and immobilised systems than in most previous studies (Table 1) Life Sciences | Biotechnology Optimisation of high productivity H pluvialis cultivation on a large-scale horizontal system produced some results Average productivity of 11.25 g m-2 d-1 and an astaxanthin content of 2.8% of the dry biomass was obtained from the m2 system in the above-described conditions (A) (B) (C) (D) Contamination was controlled during the cultivation period (Fig 8) The m2 system provided slightly lower yields than the 0.05 m2 system However, astaxanthin productivity was higher in both suspended and immobilised systems than in most previous studies (Table 1) Fig Surface of H pluvialis biofilm (A) and after 10 days of cultivation (B and C) on a m2 system; (D) Microscope image of H pluvialis after 10 days of cultivation (x40) Table Comparison of H pluvialis cultivation results on an angled biofilm-based photobioreactor system with other cultivation 11 system based on surface area Light condition (µmol photon m-2 s-1) Stess factor Cultivation period (green phase + red phase) (days) Astaxanthin content (% dried biomass) Astaxanthin productivity (mg l-1 day-1) Astaxanthin productivity (mg m-2 day-1) Dried biomass productivity References (g m-2 day-1) Intense light (Red phase) 3.6 7.2 136.8a 3.8a [33] System Strain Medium Temp (°C) Outdoor tube (50 l) Isolated BG11 25 For Sunlight controlling 400-1600 pH Outdoor open pond ZY-18 NIES-N 28 None Sunlight Max 1000 Intense light + N limited 20 (Green phase 1.7 + red phase) -/- 40a 2.34a [29] Indoor open pond 26 BG11 20 For 20-350 controlling 14/10 hour pH Intense light 12 (Green phase 2.79 + red phase) 4.3 61a 2.2a [34] Indoor bubble column ZY-18 NIES-N 28 None 250 Continuous Intense light + N limited 12 (Green phase 3.6 + red phase) -/- 237.6a 6.6a [29] Indoor bubble column (0.5 l) K-0084 Modified BG11 25 1.5 350 Continuous Intense light + N limited (Red phase) 11.5 528a 13.2a [13] Indoor closed container (10 l) HB (isolated) Modified RM 25 For 85 controlling 16/8 hour pH Intense light, N limited, high C/N, + bicarbonate 30 (green phase) 4.88 + (Red phase) 2.75 92a 1.88a [23] Indoor bubble column (5 l) -/- RM 25 40 ml/min 60 16/8 hour N limited, High C/N 22 (Green phase -/+ red phase) 0.009 0.264a -/- [24] NIES-N 25 None 150 Continuous N limited 12 (Green phase 1.3 + red phase) -/- 65.8 3.7 [28] Indoor immobilised SAG 34-1b BG11 biofilm (0.08 m2) 25 1.5 100 Continuous N limited or exhausted (Green phase + 2.2 red phase) -/- 143 6.5 [10] Indoor immobilised CCAC biofilm (0.05 m2) 0125 Modified BG11 26 650 14/10 hour Intense light (Green phase + 3.5 + N, P limited red phase) -/- 371 10.6 [32] Indoor angled CCAC immobilised biofilm 0125 (0.05 m2) Modified BG11 26 For 600-700 controlling 14/10 hour pH Intense light 10 (Green phase 3.0 + N, P limited + red phase) 7.2 360 12 This study Indoor angled CCAC immobilised biofilm 0125 (2 m2) Modified BG11 26 For 600-700 controlling 14/10 hour pH Intense light 10 (Green phase 2.8 + N, P limited + red phase) 6.3 315 11.25 This study Indoor immobilised NIES-144 biofilm (0.08 m2) a: CO2 (%) the values are converted to ‘per surface area’ 68 Vietnam Journal of Science, Technology and Engineering September 2019 • Vol.61 Number 4.0 Life Sciences | Biotechnology Conclusions Dermatol., 18(1), pp.242-250 Angled immobilised cultivation systems for H pluvialis were successfully designed and operated The dry biomass productivity and microalgal astaxanthin content of the m2 system reached 11.25 g m-2 d-1 and 2.8%, respectively, which are similar to or higher than that of other systems Both biomass and astaxanthin production can likely be improved by optimisation of the cultivation process The data show that these systems can be applied for production at a larger scale Further studies will be rewarding to improve the dry biomass and astaxanthin productivity of H pluvialis cultivated on an angled TL biofilm-based photobioreactor system Angled immobilised cultivation on the TL-biofilm-based system provides remarkable advantages compared with traditional suspended cultivation, such as in term of water, energy, and cultivation time-saving The angled system is also likely easier to scale up than the vertical TL system and perhaps more cost-efficient (for further discussion of vertical vs horizontal TL systems, see Podola, et al (2017) [35]) However, understanding the underlying processes (light, nutrient, and air distribution, etc.) in the TL system is still limited relative to suspended systems, although some progress has recently been made [36-39] ACKNOWLEDGEMENTS The authors would like to thank the support from Vietnamese Ministry of Industry and Trade for the project (03/HD-DT.03.16/CNSHCB) The authors declare that there is no conflict of interest regarding the publication of this article REFERENCES [1] R.T Lorenz, G.R Cysewski (2000), “Commercial potential for Haematococcus microalgae as a natural source of astaxanthin”, Trends in Biotechnology, 18(4), pp.160-167 [2] A.R Rao, et al (2010), “Characterization of microalgal carotenoids by mass spectrometry and their bioavailability and antioxidant properties elucidated in rat model”, J Agric Food Chem., 58(15), pp.8553-8559 [5] N Ito, S Seki, F Ueda (2018), “The protective role of astaxanthin for uv-induced skin deterioration in healthy people-a randomized, double-blind, placebo-controlled trial”, Nutrients, 10(7), Doi: 10.3390/nu10070817 [6] R.R Ambati, et al (2014), “Astaxanthin: sources, extraction, stability, biological activities and its commercial applications - a review”, Mar Drugs, 12(1), pp.128-152 [7] A.R Rao, et al (2013), “Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga H pluvialis”, J Agric Food Chem., 61(16), pp.3842-3851 [8] F Visioli, C Artaria (2017), “Astaxanthin in cardiovascular health and disease: mechanisms of action, therapeutic merits, and knowledge gaps”, Food Funct., 8(1), pp.39-63 [9] C Duk Kang, et al (2006), “Productive encystment of H pluvialis by controlling a specific irradiation rate in a photoautotrophic induction system for astaxanthin production”, J Ind Eng Chem., 12(5), pp.745-748 [10] W Zhang, et al (2014), “Attached cultivation of H pluvialis for astaxanthin production”, Bioresour Technol., 158, pp.329-335 [11] C Hagen, S Siegmund, W Braune (2002), “Ultrastructural and chemical changes in the cell wall of H pluvialis (Volvocales, Chlorophyta) during aplanospore formation”, European Journal of Phycology, 37(2), pp.217-226 [12] M Olaizola, M Huntley (2003), “Recent advances in commercial production of astaxanthin from micrsoalgae”, Recent Advances in Marine Biotechnology, 9, pp.143-164 [13] C Aflalo, et al (2007), “On the relative efficiency of two- vs one-stage production of astaxanthin by the green alga H pluvialis”, Biotechnol Bioeng., 98(1), pp.300-305 [14] I.S Suh, H.N Joo, C.G Lee (2006), “A novel doublelayered photobioreactor for simultaneous H pluvialis cell growth and astaxanthin accumulation”, J Biotechnol., 125(4), pp.540-546 [15] F.G Acién, et al (2017), “Photobioreactors for the production of microalgae”, Microalgae-based biofuels and bioproducts, pp.1-44, Woodhead Publishing [16] L Brennan, O Owende (2010), “Biofuels from microalgae - a review of technologies for production, processing, and extractions of biofuels and co-products”, Renewable and Sustainable Energy Reviews, 14(2), pp.557-577 [17] O Giuseppe, S Piero, M Antonio (2014), “Advances in photobioreactors for intensive microalgal production: configurations, operating strategies and applications”, Journal of Chemical Technology & Biotechnology, 89(2), pp.178-195 [18] M Gross, et al (2015), “Biofilm-based algal cultivation systems”, Appl Microbiol Biotechnol., 99(14), pp.5781-5789 [3] L.Y Dong, et al (2013), “Astaxanthin attenuates the apoptosis of retinal ganglion cells in db/db mice by inhibition of oxidative stress”, Mar Drugs, 11(3), pp.960-974 [19] R.A Andersen (2005), Algal culturing techniques, 596pp, Elsevier [4] B Eren, et al (2018), “Antioxidant properties evaluation of topical astaxanthin formulations as anti-aging products”, J Cosmet [20] D.H Dinh, et al (2011), “Study on cell morphology, pigment content and intracellular protein in the life cycle of H pluvialis September 2019 • Vol.61 Number Vietnam Journal of Science, Technology and Engineering 69 Life Sciences | Biotechnology cultured under laboratory conditions”, Journal of Biology, 33(1), pp.59-66 [21] D.H Dang, et al (2012), “Effect of nitrate concentration and irradiation regulation on the growth of H pluvialis”, Journal of Biology, 34(4), pp.493-499 [22] T.T Le, et al (2013), “Effect of nitrate concentration on growth of green microalga H pluvialis under laboratory conditions”, Journal of Biology, 35(2), pp.219-226 [23] T.T Luu, et al (2015), "A limiting factor for astaxanthin accumulation in phase of H pluvialis microalgae in laboratory conditions", Journal of Biotechnology, 13, pp.1-7 [24] N.N Trinh, et al (2017), “Research to improve the accumulation of astaxanthin in H pluvialis microalgae by the stress conditions of the culture medium”, Conference of the 35th anniversary of establishment of the Ho Chi Minh city University of Food Industry, pp.74-83 [25] E.C.M Nowack, B Podola, M Melkonian (2005), “The 96-well twin-layer system: a novel approach in the cultivation of microalgae”, Protist, 156(2), pp.239-251 [26] J Shi, B Podola, M Melkonian (2007), “Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study”, Journal of Applied Phycology, 19(5), pp.417-423 [27] T Naumann, et al (2013), “Growing microalgae as aquaculture feeds on twin-layers: a novel solid-state photobioreactor”, Journal of Applied Phycology, 25(5), pp.1413-1420 [28] M Wan, et al (2014a), “The effective photoinduction of H pluvialis for accumulating astaxanthin with attached cultivation”, Bioresour Technol., 163, pp.26-32 [29] M Wan, et al (2014b), “The effect of temperature on cell growth and astaxanthin accumulation of H pluvialis during a lightdark cyclic cultivation”, Bioresour Technol., 167, pp.276-283 [30] S Yin, J Wang, L Chen, T Liu (2015), “The water 70 Vietnam Journal of Science, Technology and Engineering footprint of biofilm cultivation of H pluvialis is greatly decreased by using sealed narrow chambers combined with slow aeration rate”, Biotechnol Lett., 37(9), pp.1819-1827 [31] D Zhang, et al (2016), “Dynamic modelling of H pluvialis photoinduction for astaxanthin production in both attached and suspended photobioreactors”, Algal Research, 13, pp.69-78 [32] A.C Kiperstok, et al (2017), “Biofilm cultivation of H pluvialis enables a highly productive one-phase process for astaxanthin production using high light intensities”, Algal Research, 21, pp.213-222 [33] G Torzillo, et al (2003), “Interplay between photochemical activities and pigment composition in an outdoor culture of H pluvialis during the shift from the green to red stage”, Journal of Applied Phycology, 15(2-3), pp.127-136 [34] B.Y Zhang, et al (2009), "Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process", Aquaculture, 295(3-4), pp.275-281 [35] B Podola, T Li, M Melkonian (2017), “Porous substrate bioreactors: a paradigm shift in microalgal biotechnology?”, Trends in Biotechnology, 35(2), pp.121-132 [36] T Li, et al (2016a), “Microscale profiling of photosynthesisrelated variables in a highly productive biofilm photobioreactor”, Biotechnology and Bioengineering, 113(5), pp.1046-1055 [37] T Li, et al (2016b), “Investigating dynamic processes in a porous substrate biofilm photobioreactor - a modeling approach”, Algal Research, 13, pp.30-40 [38] T Li, M Strous, M Melkonian (2017), “Biofilm-based photobioreactors: their design and improving productivity through efficient supply of dissolved inorganic carbon”, FEMS Microbiology Letters, 364(24), Doi: 10.1093/femsle/fnx218 [39] T Li, et al (2019), “Design scenario analysis for porous photobioreactor assemblies”, Journal of Applied Phycology, 31(3), pp.1623-1636 September 2019 • Vol.61 Number ... protocol for immobilised H pluvialis cultivation Cultivation of H pluvialis in the astaxanthin accumulation phase onhigh an productivity angled Immobilised algae cultivation for astaxanthin harvest on. .. Nutrient and air supply system for cultivation chamber of bench-scale system; (B) Positioning of chambers and lights in Nutrient and for cultivation chamber of bench-scale Fig system;3 (C) (A) The bench-scale. .. suspended cultivation of H pluvialis is more common for the production of astaxanthin at the commercial scale Suspended cultivation is applied in open ponds or closed photobioreactors Open-pond cultivation